The incorporation of [3H]fucose into the novel fucosides glucosyl-fucosyl-threonine(FL4a) and fucosyl-threonine(FL3a) has been utilized in this laboratory and by others to predict the tumorigenic and metastatic potential of transformed rat fibroblasts and transformed mouse epithelial cells. During the past year, we have continued our studies of the chemical and metabolic characterization of the parent glycoproteins of FL4a and FL3a. Approximately 90% of the putatively identified parent proteins of FL4a were in the membrane fraction, and a substantial proportion were released into the medium as a function of growth time. For example, after 96 hrs of labeling there were 6-fold more of these components in the growth medium than were cell-associated. Moreover, the incorporation of labeled fucose into the parent glycoproteins appeared to be cell population density-dependent. When the distribution of these compounds was compared in normal versus cancer cells, it was found that a higher proportion of the proteins with O-linked fucose were shed into the medium of the cancer cells. The enhanced shedding could explain the basis for the reduced level of FL4a in cancer cells, since the metabolic labeling experiments did not indicate a marked difference in the metabolism of either the parent protein or in FL4a or FL3a. Despite the apparent wide distribution of these novel fucose substituents in cellular proteins, it seems reasonable to suggest that they have not been routinely observed largely because each represents less than 0.5% of the fucose bound to protein. (A)